1. Field of the Invention
The present invention relates to an acetyltransferase gene-containing DNA. More particularly, the present invention is concerned with a DNA comprising a gene coding for deacetylcephalosporin C acetyltransferase which is an enzyme capable of catalyzing the final step of the biosynthesis of cephalosporin C. The gene may be either a cDNA or a genomic DNA. The present invention also relates to a recombinant DNA comprising a vector having inserted therein a DNA coding for at least a portion of the above-mentioned gene and being capable of expression in Acremonium chrysogenum, and relates to Acremonium chrysogenum transformed with the recombinant DNA. Furthermore, the present invention relates to a method for preparing cephalosporin C by culturing the transformed Acremonium chrysogenum. By this method, cephalosporin C can be prepared efficiently.
2. Discussion of Related Art
The biosynthesis of cephalosporin C proceeds according to the following sequence of reactions: ##STR1##
In the art, an enzyme capable of catalyzing the reaction of step 2, i.e., isopenicillin N synthetase, and enzymes capable of catalyzing the reactions of steps 3 and 4, i.e., deacetoxycephalosporin C synthetase and deacetylcephalosporin C synthetase were isolated from Acremonium chrysogenum. It is known that in Acremonium chrysogenum, a single polypeptide has both of the two enzymatic activities necessary for steps 3 and 4. Also, the genes coding for such enzymes were isolated, and the nucleotide sequences of the genes were identified. With respect to the above, reference is made to for example, Samson et al., Nature (1985) 318, 191; Dotzlaf et al., Journal of Bacteriology (1987) 169, 1611-1618; Samson et al., Biotechnology (1987) i, 1207; and Japanese Patent Application laid-Open Specification No. 63-301790.
Skatrud et al. noted that a significant amount of penicillin N, i.e., an intermediate of the above-mentioned biosynthesis of cephalosporin C, was present in the medium in which Acremonium chrysogenum was fermented, and introduced a vector containing a DNA fragment carrying a gene coding for deacetoxycephalosporin C synthetase/deacetylcephalosporin C synthetase (hereinafter often referred to as "DAOCS/DACS"), penicillin N being a substrate thereof, into Acremonium chrysogenum. As a result, Skatrud et al. succeeded in increasing the level of DAOCS/DACS in the cells and thus improving the yield of cephalosporin C [see Skatrud et al., Bio/technology (1989) 7, 477-485].
On the other hand, it was known in the art that in the above-mentioned medium in which Acremonium chrysogenum was fermented, a significant amount of deacetylcephalosporin C, i.e., another intermediate of the biosynthesis of cephalosporin C, was also present [see Huber et al., Applied Microbiology (1968) 16., 1011-1014]. Accordingly, an increase in the intracellular level of an enzyme capable of catalyzing the conversion of the above-mentioned intermediate to cephalosporin C (i.e., step 6 of the biosynthesis of cephalosporin C), namely, deacetylcephalosporin C acetyltransferase and a modification of the enzyme so as to be more suitable for the preparation of cephalosporin C would improve the yield of cephalosporin C.
For the above reason, the purification of deacetylcephalosporin C acetyltranserase and the isolation of a gene coding for the enzyme were strongly desired in the art.
The present inventors succeeded in isolating deacetylcephalosporin C acetyltransferase from Acremonium chrysogenum, and clarified that the enzyme was comprised of at least two subunits (the subunit having a molecular weight of 27,000.+-.2000 dalton as measured by SDS polyacrylamide gel electrophoresis was designated as subunit 1 while the subunit having a molecular weight of 14,000.+-.2000 dalton as measured by the above-mentioned electrophoresis was designated as subunit 2). Further, the present inventors determined the amino acid sequence in N-terminus portion of each of subunit 1 and subunit 2, which was as follows.
______________________________________ (subunit 1) Leu--X--Ala--Gln--Asp--Ile--Ala--Arg--Ile-- Ser--leu--Phe--Thr--Leu--Glu--Ser--Gly-- Val--Ile--Leu--Arg (wherein X indicates a site where identification of an amino acid was not made) (subunit 2) Asp--Ser--Gly--Asn--Ser--His--Arg--Ala-- Gly--Gln--Pro--Ile--Glu--Ala--Val--Ser-- Ser--Tyr--Leu--Arg--Tyr--Gln--Ala--Gln-- Lys--Phe--Ala ______________________________________
Despite various developments as mentioned above, isolation of a gene (genomic DNA) of deacetylcephalosporin C acetyltransferase has not yet been attained in the art.